The human malaria parasite Plasmodium falciparum is responsible for 300-500 million cases of malaria and 1.3 million deaths every year. The recent emergence and spread of resistance to many current antimalarial drugs stimulated efforts to develop new compounds to prevent and treat malaria. Completion of the P. falciparum genome sequence six years ago provided many novel and unexpected insights into malaria parasite metabolism that led to the identification of new drug targets. Using comparative genome analyses, we recently identified P. falciparum nuclear genes encoding apicoplast-targeted orthologs of glutamyl-tRNA synthetase and glutamyl-tRNA amidotransferase, two enzymes that constitute an indirect aminoacylation pathway for the production of Gln-tRNAGln, a key substrate required for protein biosynthesis. This pathway is the sole route for the production of Gln-tRNAGln in most bacteria, archaea, and plastids, but is not found in the eukaryotic cytosol. Inhibitors of the second enzyme in the pathway, glutamyl-tRNA amidotransferase, possess anti-bacterial activity but are not toxic to mammalian cells, indicating that the Plasmodium enzyme may be a good target for drug therapy. Apicoplast-targeting of two subunits of P. falciparum glutamyl-tRNA amidotransferase was detected by transfection of parasites with episomal constructs encoding the putative apicoplast leader sequences fused to green fluorescent protein. In addition, recombinant apicoplast glutamyl-tRNA synthetase expressed in wheat germ exhibited both discriminating and non-discriminating activity by glutamylating apicoplast tRNAGlu and tRNAGln, an essential property of tRNA synthetases that participate in indirect aminoacylation pathways in bacteria and plastids. Together, these data strongly support the hypothesis that biosynthesis of Gln-tRNAGln in the Plasmodium apicoplast occurs via indirect rather than direct aminoacylation. The proposed studies will use molecular genetic and biochemical approaches to characterize both of the Plasmodium enzymes, identify their tRNA substrates, determine their subcellular localization, and evaluate the sensitivity of Plasmodium glutamyl-tRNA amidotransferase to inhibitors of the bacterial enzyme. Completion of these goals will demonstrate that the proposed indirect aminoacylation pathway in the apicoplast is functional and set the stage for subsequent studies to validate indirect aminoacylation in the apicoplast as a target for malaria chemotherapy. PUBLIC HEALTH RELEVANCE: The human malaria parasite Plasmodium falciparum causes over 300 million cases of malaria and 1.3 million deaths annually. New drugs are required to treat and prevent malaria because of resistance to existing anti-malarial drugs. This research program will characterize a novel Plasmodium biochemical pathway that we believe is essential to parasite survival and therefore a potential target for drug therapy of malaria.